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Acta Biochimica Et Biophysica Sinica May 2022Cartilage and subchondral bone communicate with each other through material and signal exchanges. However, direct evidence provided by experimental studies on their...
Cartilage and subchondral bone communicate with each other through material and signal exchanges. However, direct evidence provided by experimental studies on their interactions is insufficient. In the present study, we establish a noncontact co-culture model with a transwell chamber to explore the energetic perturbations in chondrocytes influenced by osteoblasts. Our results indicate that osteoblasts induce more ATP generation in chondrocytes through an energetic shift characterized by enhanced glycolysis and impaired mitochondrial tricarboxylic acid cycle. Enhanced glycolysis is shown by an increase of secreted lactate and the upregulation of glycolytic enzymes, including glucose-6-phosphate isomerase (Gpi), liver type ATP-dependent 6-phosphofructokinase (Pfkl), fructose-bisphosphate aldolase C (Aldoc), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), triosephosphate isomerase (Tpi1), and phosphoglycerate kinase 1 (Pgk1). Impaired mitochondrial tricarboxylic acid cycle is characterized by the downregulation of cytoplasmic aspartate aminotransferase (Got1) and mitochondrial citrate synthase (Cs). Osteoblasts induce the activation of Akt and P38 signaling to mediate ATP perturbations in chondrocytes. This study may deepen our understanding of the maintenance of metabolic homeostasis in the bone-cartilage unit.
Topics: Glucose-6-Phosphate Isomerase; Fructose-Bisphosphate Aldolase; Triose-Phosphate Isomerase; Chondrocytes; Glucose; Aspartate Aminotransferase, Cytoplasmic; Phosphoglycerate Kinase; Proto-Oncogene Proteins c-akt; Citrate (si)-Synthase; Glycolysis; Glyceraldehyde-3-Phosphate Dehydrogenases; Phosphofructokinase-1; Osteoblasts; Communication; Lactates; Adenosine Triphosphate
PubMed: 35593470
DOI: 10.3724/abbs.2022042 -
Microbiology (Reading, England) Nov 2017In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of...
In (hyper)thermophilic organisms metabolic processes have to be adapted to function optimally at high temperature. We compared the gluconeogenic conversion of 3-phosphoglycerate via 1,3-bisphosphoglycerate to glyceraldehyde-3-phosphate at 30 °C and at 70 °C. At 30 °C it was possible to produce 1,3-bisphosphoglycerate from 3-phosphoglycerate with phosphoglycerate kinase, but at 70 °C, 1,3-bisphosphoglycerate was dephosphorylated rapidly to 3-phosphoglycerate, effectively turning the phosphoglycerate kinase into a futile cycle. When phosphoglycerate kinase was incubated together with glyceraldehyde 3-phosphate dehydrogenase it was possible to convert 3-phosphoglycerate to glyceraldehyde 3-phosphate, both at 30 °C and at 70 °C, however, at 70 °C only low concentrations of product were observed due to thermal instability of glyceraldehyde 3-phosphate. Thus, thermolabile intermediates challenge central metabolic reactions and require special adaptation strategies for life at high temperature.
Topics: Enzyme Stability; Gluconeogenesis; Glyceraldehyde 3-Phosphate; Glyceraldehyde-3-Phosphate Dehydrogenases; Glyceric Acids; Half-Life; Hot Temperature; Kinetics; Models, Statistical; Phosphoglycerate Kinase; Recombinant Proteins; Saccharomyces cerevisiae; Substrate Cycling; Sulfolobus solfataricus; Thermodynamics
PubMed: 28982396
DOI: 10.1099/mic.0.000542 -
International Journal of Molecular... Dec 2021Gastrointestinal disease is the most common health concern that occurs due to environmental, infectious, immunological, psychological, and genetic stress. Among them,...
Gastrointestinal disease is the most common health concern that occurs due to environmental, infectious, immunological, psychological, and genetic stress. Among them, the most frequent diseases are gastric ulcer (GU) and ulcerative colitis (UC). DSS-induced UC and ethanol-stimulated GU models resemble the pathophysiology of human gastrointestinal disease. The current study was designed to explore the anti-oxidation, anti-inflammation, anti-cell death properties of terazosin, an α-adrenergic receptor antagonist, in vivo and in vitro. Our results indicate that terazosin dramatically activates Pgk1, and upregulates glycose metabolism, evidenced by the enhanced ATP production and higher LDH enzymatic activity. Also, terazosin significantly enhances p-AKT expression and inhibits NF-κB p65 activation through abrogating the phosphorylation of IKBα, as well as lowers Caspase-1 and GSDMD expression. The findings in this study demonstrate that terazosin exhibits anti-inflammatory effects by downregulating NF-κB-GSDMD signal pathway, along with enhancing glycolysis for gastrointestinal disease treatment. Meanwhile, we also find terazosin ameliorates ethanol-induced gastric mucosal damage in mice. Collectively, as a clinical drug, terazosin should be translated into therapeutics for gastrointestinal disease soon.
Topics: Apoptosis; Caco-2 Cells; Cell Survival; Colitis; Cytokines; Deoxyglucose; Dextran Sulfate; Gastric Mucosa; Gastrointestinal Diseases; Glucose; Humans; Hydrogen Peroxide; Inflammation Mediators; Lactic Acid; Malondialdehyde; Models, Biological; Peroxidase; Phosphoglycerate Kinase; Prazosin; Pyroptosis; Stomach Ulcer; Superoxide Dismutase
PubMed: 35008842
DOI: 10.3390/ijms23010416 -
Perturbation of phosphoglycerate kinase 1 (PGK1) only marginally affects glycolysis in cancer cells.The Journal of Biological Chemistry May 2020Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell...
Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 μm (yeast PGK1) and 6.86 μm (human PKG1). The values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 μm (yeast PGK1) and 186 μm (human PGK1). The of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 μm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis.
Topics: A549 Cells; Diphosphoglyceric Acids; Glucose; Glyceric Acids; Glycolysis; HeLa Cells; Humans; Kinetics; Lactic Acid; Neoplasm Proteins; Neoplasms; Phosphoglycerate Kinase; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 32217690
DOI: 10.1074/jbc.RA119.012312 -
Plant Physiology Feb 2018In plants, phosphoglycerate kinase (PGK) converts 1,3-bisphosphoglycerate into 3-phosphoglycerate in glycolysis but also participates in the reverse reaction in...
In plants, phosphoglycerate kinase (PGK) converts 1,3-bisphosphoglycerate into 3-phosphoglycerate in glycolysis but also participates in the reverse reaction in gluconeogenesis and the Calvin-Benson cycle. In the databases, we found three genes that encode putative PGKs. Arabidopsis () PGK1 was localized exclusively in the chloroplasts of photosynthetic tissues, while PGK2 was expressed in the chloroplast/plastid of photosynthetic and nonphotosynthetic cells. PGK3 was expressed ubiquitously in the cytosol of all studied cell types. Measurements of carbohydrate content and photosynthetic activities in PGK mutants and silenced lines corroborated that PGK1 was the photosynthetic isoform, while PGK2 and PGK3 were the plastidial and cytosolic glycolytic isoforms, respectively. The knockdown mutant displayed reduced growth, lower photosynthetic capacity, and starch content. The knockout mutant was characterized by reduced growth but higher starch levels than the wild type. The double mutant was bigger than and displayed an intermediate phenotype between the two single mutants in all measured biochemical and physiological parameters. Expression studies in mutants showed that and were down-regulated in and , respectively. These results indicate that the down-regulation of photosynthetic activity could be a plant strategy when glycolysis is impaired to achieve metabolic adjustment and optimize growth. The double mutants of and the triose-phosphate transporter ( displayed a drastic growth phenotype, but they were viable. This implies that other enzymes or nonspecific chloroplast transporters could provide 3-phosphoglycerate to the cytosol. Our results highlight both the complexity and the plasticity of the plant primary metabolic network.
Topics: Arabidopsis; Arabidopsis Proteins; Cytosol; Gene Expression Regulation, Plant; Glyceric Acids; Metabolomics; Multigene Family; Mutation; Phosphoglycerate Kinase; Plant Components, Aerial; Plant Roots; Plants, Genetically Modified; Plastics
PubMed: 28951489
DOI: 10.1104/pp.17.01227 -
Biomacromolecules Oct 2022Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely...
Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely established. Alginate is widely used to encapsulate proteins for drug delivery and tissue regeneration despite limited knowledge of its impact on protein stability. Here, we present evidence that alginate can both increase protein folding stability and suppress the aggregation of unfolded protein through direct interactions without crowding. We used a fluorescence-based conformational reporter of two proteins, the metabolic protein phosphoglycerate kinase (PGK) and the hPin1 WW domain to monitor protein stability and aggregation as a function of temperature and the weight percent of alginate in solution. Alginate stabilizes PGK by up to 14.5 °C, but stabilization is highly protein-dependent, and the much smaller WW domain is stabilized by only 3.5 °C against thermal denaturation. Stabilization is greatest at low alginate weight percent and decreases at higher alginate concentrations. This trend cannot be explained by crowding, and ionic screening suggests that alginate stabilizes proteins through direct interactions with a significant electrostatic component. Alginate also strongly suppresses aggregation at high temperature by irreversibly associating with unfolded proteins and preventing refolding. Both the beneficial and negative impacts of alginate on protein stability and aggregation have important implications for practical applications.
Topics: Alginates; Phosphoglycerate Kinase; Polymers; Protein Denaturation; Protein Folding; Protein Stability
PubMed: 36054903
DOI: 10.1021/acs.biomac.2c00297 -
Cell Death & Disease Feb 2023Glycolysis is the most predominant metabolic reprogramming of pancreatic cancer (PC), the underlying mechanism of which in PC cells remains unclear. In this study, we...
Glycolysis is the most predominant metabolic reprogramming of pancreatic cancer (PC), the underlying mechanism of which in PC cells remains unclear. In this study, we found for the first time that KIF15 promotes the glycolytic capacity of PC cells and PC tumor growth. Moreover, the expression of KIF15 was negatively correlated with the prognosis of PC patients. The ECAR and OCR measurements indicated that KIF15 knockdown significantly impaired the glycolytic capacity of PC cells. Western blotting demonstrated that the expression of glycolysis molecular markers decreased rapidly after the knockdown of KIF15. Further experiments revealed that KIF15 promoted the stability of PGK1 and its effect on PC cell glycolysis. Interestingly, the overexpression of KIF15 impaired the ubiquitination level of PGK1. To investigate the underlying mechanism by which KIF15 regulates the function of PGK1, we performed mass spectrometry (MS). The MS and Co-IP assay indicated that KIF15 recruited and enhanced the binding between PGK1 and USP10. The ubiquitination assay verified that KIF15 recruited and promoted the effect of USP10 on PGK1, thereby deubiquitinating PGK1. Through the construction of KIF15 truncators, we found that KIF15 is bound to PGK1 and USP10 through its coil2 domain. Together, our study demonstrated for the first time that KIF15 enhances the glycolytic capacity of PC through the recruitment of USP10 and PGK1, and that the KIF15/USP10/PGK1 axis may serve as an effective therapeutic agent for PC.
Topics: Humans; Pancreatic Neoplasms; Ubiquitination; Glycolysis; Cell Line, Tumor; Cell Proliferation; Kinesins; Ubiquitin Thiolesterase; Phosphoglycerate Kinase
PubMed: 36807568
DOI: 10.1038/s41419-023-05679-2 -
Protein Science : a Publication of the... Apr 2020Ficoll, an inert macromolecule, is a common in vitro crowder, but by itself it does not reproduce in-cell stability or kinetic trends for protein folding. Lysis buffer,...
Ficoll, an inert macromolecule, is a common in vitro crowder, but by itself it does not reproduce in-cell stability or kinetic trends for protein folding. Lysis buffer, which contains ions, glycerol as a simple kosmotrope, and mimics small crowders with hydrophilic/hydrophobic patches, can reproduce sticking trends observed in cells but not the crowding. We previously suggested that the proper combination of Ficoll and lysis buffer could reproduce the opposite in-cell folding stability trend of two proteins: variable major protein-like sequence expressed (VlsE) is destabilized in eukaryotic cells and phosphoglycerate kinase (PGK) is stabilized. Here, to discover a well-characterized solvation environment that mimics in-cell stabilities for these two very differently behaved proteins, we conduct a two-dimensional scan of Ficoll (0-250 mg/ml) and lysis buffer (0-75%) mixtures. Contrary to our previous expectation, we show that mixtures of Ficoll and lysis buffer have a significant nonadditive effect on the folding stability. Lysis buffer enhances the stabilizing effect of Ficoll on PGK and inhibits the stabilizing effect of Ficoll on VlsE. We demonstrate that a combination of 150 mg/ml Ficoll and 60% lysis buffer can be used as an in vitro mimic to account for both crowding and non-steric effects on PGK and VlsE stability and folding kinetics in the cell. Our results also suggest that this mixture is close to the point where phase separation will occur. The simple mixture proposed here, based on commercially available reagents, could be a useful tool to study a variety of cytoplasmic protein interactions, such as folding, binding and assembly, and enzymatic reactions. SIGNIFICANCE STATEMENT: The complexity of the in-cell environment is difficult to reproduce in the test tube. Here we validate a mimic of cellular crowding and sticking interactions in a test tube using two proteins that are differently impacted by the cell: one is stabilized and the other is destabilized. This mimic is a starting point to reproduce cellular effects on a variety of protein and biomolecular interactions, such as folding and binding.
Topics: Biomimetics; Eukaryotic Cells; Fluorescence Resonance Energy Transfer; Humans; In Vitro Techniques; Kinetics; Models, Molecular; Protein Folding; Proteins; Solubility; Thermodynamics; Tumor Cells, Cultured
PubMed: 31994240
DOI: 10.1002/pro.3833 -
Angewandte Chemie (International Ed. in... Apr 2017The 1994 structure of a transition-state analogue with AlF and GDP complexed to G1α, a small G protein, heralded a new field of research into the structure and... (Review)
Review
The 1994 structure of a transition-state analogue with AlF and GDP complexed to G1α, a small G protein, heralded a new field of research into the structure and mechanism of enzymes that manipulate the transfer of phosphoryl (PO ) groups. The number of enzyme structures in the PDB containing metal fluorides (MF ) as ligands that imitate either a phosphoryl or a phosphate group was 357 at the end of 2016. They fall into three distinct geometrical classes: 1) Tetrahedral complexes based on BeF that mimic ground-state phosphates; 2) octahedral complexes, primarily based on AlF , which mimic "in-line" anionic transition states for phosphoryl transfer; and 3) trigonal bipyramidal complexes, represented by MgF and putative AlF moieties, which mimic the geometry of the transition state. The interpretation of these structures provides a deeper mechanistic understanding into the behavior and manipulation of phosphate monoesters in molecular biology. This Review provides a comprehensive overview of these structures, their uses, and their computational development.
Topics: Aluminum Compounds; Animals; Fluorides; Humans; Models, Molecular; Phosphates; Phosphoglycerate Kinase; Phosphoric Monoester Hydrolases
PubMed: 27862756
DOI: 10.1002/anie.201606474 -
Experimental Parasitology Aug 2023Per-ARNT-Sim (PAS) domains constitute a family of domains present in a wide variety of prokaryotic and eukaryotic organisms. They form part of the structure of various...
Per-ARNT-Sim (PAS) domains constitute a family of domains present in a wide variety of prokaryotic and eukaryotic organisms. They form part of the structure of various proteins involved in diverse cellular processes. Regulation of enzymatic activity and adaptation to environmental conditions, by binding small ligands, are the main functions attributed to PAS-containing proteins. Recently, genes for a diverse set of proteins with a PAS domain were identified in the genomes of several protists belonging to the group of kinetoplastids, however, until now few of these proteins have been characterized. In this work, we characterize a phosphoglycerate kinase containing a PAS domain present in Trypanosoma cruzi (TcPAS-PGK). This PGK isoform is an active enzyme of 58 kDa with a PAS domain located at its N-terminal end. We identified the protein's localization within glycosomes of the epimastigote form of the parasite by differential centrifugation and selective permeabilization of its membranes with digitonin, as well as in an enriched mitochondrial fraction. Heterologous expression systems were developed for the protein with the N-terminal PAS domain (PAS-PGKc) and without it (PAS-PGKt), and the substrate affinities of both forms of the protein were determined. The enzyme does not exhibit standard Michaelis-Menten kinetics. When evaluating the dependence of the specific activity of the recombinant PAS-PGK on the concentration of its substrates 3-phosphoglycerate (3PGA) and ATP, two peaks of maximal activity were found for the complete enzyme with the PAS domain and a single peak for the enzyme without the domain. Km values measured for 3PGA were 219 ± 26 and 8.8 ± 1.3 μM, and for ATP 291 ± 15 and 38 ± 2.2 μM, for the first peak of PAS-PGKc and for PAS-PGKt, respectively, whereas for the second PAS-PGKc peak values of approximately 1.1-1.2 mM were estimated for both substrates. Both recombinant proteins show inhibition by high concentrations of their substrates, ATP and 3PGA. The presence of hemin and FAD exerts a stimulatory effect on PAS-PGKc, increasing the specific activity by up to 55%. This stimulation is not observed in the absence of the PAS domain. It strongly suggests that the PAS domain has an important function in vivo in T. cruzi in the modulation of the catalytic activity of this PGK isoform. In addition, the PAS-PGK through its PAS and PGK domains could act as a sensor for intracellular conditions in the parasite to adjust its intermediary metabolism.
Topics: Humans; Trypanosoma cruzi; Phosphoglycerate Kinase; Protein Isoforms; Chagas Disease; Adenosine Triphosphate
PubMed: 37353138
DOI: 10.1016/j.exppara.2023.108574